Various kinds of viruses exist on the earth, and some of them are pathogens. For example, it is known that a hepatitis virus as a cause of viral hepatitis is transmissible at the time of blood transfusion, injection, childbirth, etc.
The blood transfusion is one of the important measures for maintaining the life of a human being. However, there have heretofore been reported a large number of cases such that the blood transfusion causes viral hepatitis. In general, the viral hepatitis is classified into hepatitis A, hepatitis B and non-A non-B hepatitis.
Conventionally, it is known that hepatitis B virus (HBV) also causes viral hepatitis at the time of the blood transfusion. In recent years, a diagnostic reagent for detecting the presence of the HBV has been developed, and it has become possible to easily confirm the presence of the HBV prior to the blood transfusion. Accordingly, the HBV infection of a human being due to the blood transfusion has entirely been prevented. On the other hand, it is also known the existence of another hepatitis (non-A non-B hepatitis) which is clearly viral, but is different from the hepatitis A caused by hepatitis A virus (HAV) and also different from the hepatitis B caused by HBV, while the HAV and HBV have heretofore been considered as the causes of hepatitis. However, it has heretofore been considered to be difficult to confirm the cause (virus) of such non-A non-B hepatitis.
In 1989, Choo, Q-L. et al. (Science, 244, 359-362, 1989) and Kuo ,G. et al. (Science, 244, 362-364, 1989) proved the existence of the non-A non-B hepatitis virus. This hepatitis virus was named "HCV" (hepatitis C virus). It has heretofore been reported that the HCV is selectively transmissible to a human being and a chimpanzee.
The above-mentioned paper describes the following experiments. That is, a preparation of blood coagulation Factor VIII which was the same as the preparation which had caused the non-A non-B hepatitis when administered to a patient, was administered to a Chimpanzee-No. 1 thereby to cause non-A non-B hepatitis. Further, a preparation extracted from the liver of the above Chimpanzee-No. 1 was similarly administered to a Chimpanzee-No. 2 thereby to cause non-A non-B hepatitis, and thereafter, the blood plasma of this Chimpanzee-No. 2 was extracted. The resultant blood plasma of the Chimpanzee-No. 2 was further administered to a Chimpanzee-No. 3, whereby the occurrence of non-A non-B hepatitis was confirmed. The resultant blood plasma which had confirmedly cause the disease, was subjected to a concentration procedure for virus particles (Bradley, D. W. et al.; Gastroenterology, 88: 773-779, 1989) under the assumption that the virus to be obtained was flavivirus, whereby RNA of the virus was extracted. Then, cDNA was synthesized on the basis of the thus obtained RNA, and a .lambda.gt11 library was prepared. With respect to the resultant .lambda.gt11 library, immunoscreening was conducted by using the blood serum of a convalescent chimpanzee or the blood serum of a chronic non-A non-B hepatitis patient, thereby to select a reactive clone. As a result, it was confirmed that only the clone named "5-1-1" was a cDNA fragment originated from HCV.
The above-mentioned procedure is also described in Japanese Patent Publication (KOKOKU) No. Hei 2-500880 (500880/1990), wherein the genetic sequence thereof is also shown. In addition, a genetic sequence of the HCV was also reported in Japan (Journal of Virology, 65(3), 1105-1113, 1991).
In the meeting of ACTA HEPATOLOGICA JAPONICA in June of 1990, a genetic sequence of the HCV structural region was shown by Okamoto of Jichi Medical School et al. (Proceedings of the 26th Meeting of ACTA HEPATOLOGICA JAPONICA in 1990). In the meeting of Japan Cancer Society in July of 1990, a genetic sequence of the HCV structural region was shown (Proceedings of the 49th meeting of Japan Cancer Society in 1990) similarly as in the above-mentioned publication by Okamoto et al. When these publications are compared with each other, it has been found that substantially no mutation is observed in the HCV core region, but a somewhat difference is recognized between these publications with respect to the HCV envelope region.
Thereafter, there has been proposed an antibody diagnosis using the core antigen of the HCV structural region in combination with an antigenic site of the HCV non-structural region (Rinsho-Byori (The Japanese Journal of Clinical Pathology), 40(12), 1245-1251, 1992). When such an antibody diagnosis is used, it has become possible to conduct an almost perfect screening for an HCV antibody-detected person.
On the other hand, in parallel with the above HCV diagnosis, a treatment for hepatitis C using interferon has been initiated. In this treatment, it has been found that the effect of the interferon treatment varies according to the kind of the genetic type (genotype) of the HCV.
The classification of the genotype has been proposed by Okamoto H. in KAN TAN SUI, 24, 7-14 (1992), and the genotypes are classified into Type-I (Proc. Natl. Acad. Sci. USA, 88, 2451-2455, 1991), Type-II (Journal of Virology, 65(3), 1105-1113, 1991), Type-III (Journal of General Virology, 72, 2697-2704, 1991), and Type-IV (Virology, 188, 331-341, 1992). In the meeting of ACTA HEPATOLOGICA JAPONICA held in June of 1993, it was reported that the interferon treatment for genotype-I and -II (interferon treatment: effective in 20% of individuals tested) was lower than that for genotype-III and -IV (interferon treatment: effective in 80% of individuals tested) (Proceedings of the 29th Meeting of ACTA HEPATOLOGICA JAPONICA, page 55, 1993).
In many cases, the administration of the interferon produces a strong side effect such as alopecia and fever, and the load thereof to the patient is heavy. Therefore, the determination of the HCV genotype prior to the treatment is extremely important in view of the prediction of the effect of the interferon administration. However, in the conventional method of determining the genotype, the HCV RNA is extracted from the specimen of a patient, cDNA complementary to the resultant RNA is synthesized, the cDNA is amplified by a PCR (polymerase chain reaction) method, and a band corresponding to the thus amplified cDNA is identified by use of electrophoresis, thereby to determine the genotype. Since such a procedure includes complicated steps and usually lasts for 48 hours or more, it requires a long period of time and a high cost.
On the other hand, it has been reported that the HCV antibodies in the blood sera of patients can be classified into two types by using an antigen (including about 300 amino acids) which has been prepared by a genetic recombination technique (KAN TAN SUI, 22, 883-889, 1991). However, this classification is not necessarily sufficient in view of the correspondence thereof with the above-mentioned genotype, which is important in determining the interferon treatment.
As described above, the administration of the interferon requires a high cost, and further, the determination of the above-mentioned genotype, which is a prerequisite for the interferon administration, has a disadvantage such that the determination per se also requires a high cost. If the genotype of the HCV can be determined easily prior to the interferon administration, it becomes possible to easily predict the effect of the interferon administration so as to properly design the method of administrating the interferon and a guideline for the treatment using the same. In addition, it becomes possible to reduce the mental, physical and economic load to the patient. Accordingly, there has eagerly been desired a simple method of determining the HCV genotype.
Accordingly, an object of the present invention is to provide an antigen for measuring an HCV antibody, which enables simple determination of the HCV antibody wherein the genotype-I and genotype-II can be distinguished from the genotype-III and genotype-IV.